Method for specific silencing of genes by DNA methylation

ABSTRACT

The present invention provides a method for the silencing of specific genes by DNA methylation. The method involves introducing into a cell a single stranded oligonucleotide containing 5-methyl deoxycytosine. The single stranded oligonucleotide has a sequence complementary to a portion of the DNA sequence of the gene to be silenced.

This application is a continuation of application Ser. No. 08/553,587filed Feb. 5, 1996, and now abandoned, which is a 371 of PCT/AU94/00314filed Jun. 10, 1994.

The present invention relates to a method for the silencing of specificgenes by DNA methylation. The method involves the introduction intocells of oligonucleotides containing 5-methyl cytosine residues, theoligonucleotide having a sequence complementary to a portion of the DNAof the gene to be silenced.

It is known that the methylation of cytosine in DNA by the enzyme DNAmethyl transferase results in the inactivation of gene expression inmany biological contexts. Furthermore, once a cytosine residue ismethylated in a CpG doublet, the enzyme recognises this substrate afterDNA replication and methylates the new strand, whereas non-methylatedCpG doublets remain unmethylated. Thus a given pattern of DNAmethylation is heritable, and the methylated genes remain silent.Non-specific gene silencing in CHO cells has been achieved by uptake of5-methyl deoxycytosine triphosphate (Holliday & Ho, 1991, Somatic Celland Molecular Genetics, 17:537-542; Nyce, 1991, Somatic Cell andMolecular Genetics, 17:543-550). The new method developed by the presentinventors can silence specific genes, provided the sequence of bases inthe promoter region is known. The method developed by the presentinventors involves the use of single stranded oligonucleotidescontaining 5-methyl deoxycytosine in appropriate CpG doublets.

Accordingly, in a first aspect the present invention consists in amethod of silencing a specific gene in a cell comprising introducinginto the cell a single stranded oligonucleotide containing 5-methyldeoxycytosine, the single stranded oligonucleotide having a sequencecomplementary to a portion of the DNA sequence of the gene to besilenced.

In a preferred embodiment of the present invention the single strandedoligonucleotide has a sequence complementary to a sequence within thepromoter region of the gene to be silenced or within CpG islands of thegene to be silenced.

As will be readily appreciated provided some or all of the cells areactively synthesising DNA (S phase), the methylated oligonucleotide cananneal or hybridize to single stranded DNA at the replication fork.Accordingly, it is presently preferred that the cells are synchronizedand treated during DNA synthesis. Synchrony can be achieved by serumstarvation, followed by addition of serum which stimulates DNAsynthesis, or other methods of synchronization.

As is clear from the above discussion the method of the presentinvention depends on the synthesis of single stranded oligonucleotidescontaining 5-methyl deoxycytosine in appropriate CpG doublets or atother sites. The synthesis is a standard procedure substituting 5-methyldeoxycytosine phosphoroamidate for cytosine phosphoroamidate (ABIsystems oligonucleotide synthesiser). The target gene is unmethylated inthe promoter region and the gene is, or can be, expressed in the cellsto be treated. The oligonucleotide can be introduced into the cells byelectroporation, Transfectam or by any other means of permealizing cellsor by other methods of introducing DNA into cells. Provided some or allof the cells are actively synthesising DNA (S phase), the methylatedoligonucleotide can anneal or hybridize to single stranded DNA at thereplication fork. This creates a hemimethylated substrate for the DNAmethyltranferase. The enzyme methylates the cellular DNA in the regionof hybridization. This methylation imprint in a short region of thepromoter silences the specific gene, and the effect is permanent becausethe methylation is subsequently inherited. In order that the target geneis specifically recognize the oligonucleotide is of sufficient length(20-40 bases) to hybridize only with this gene and no other in thegenome. Alternatively, the oligonucleotide could hybridise to the geneduring active transcription, because there is some unwinding of the DNAduring this process. The end result is the same.

The cells can be primary human cells, or permanent lines of human oranimal origin. The cells should be synchronized and treated during DNAsynthesis. Synchrony can be achieved by serum starvation, followed byaddition of serum which stimulates DNA synthesis, or any alternativemethod of synchronization can be used.

To detect gene silencing it is desirable or necessary to have aselection procedure which eliminates cells containing the active gene.Many such selection procedures are available including genes whichsynthesise thymidine kinase, adenine phosphoribosyl transferase andhypoxanthine phosphoribosyl tranferase. There should be only one activecopy of the gene to be targeted. Genes of the histocompatibility complexsuch as HLA-A in human cells produce a cell surface antigen. Anappropriate antiserum and complement will kill all cells expressing theantigen. In this case the gene is often heterozygous (e.g.HLA-A2/HLA-A3) and it is possible to select cells which are notexpressing either haplotype (e.g. by using antiserum against A2 or A3antigens). Although the promoter region of both genes may be the same,the methylation of one will eliminate the gene product and such cellscan be selected. These can be used in a second treatment with methylatedoligonucleotide which can yield cells lacking both surface antigens.

Many inherited human diseases are caused by dominant genes. These mayproduce an excess of a gene product or an altered gene product. Cellsfrom such individuals could be made normal if the dominant gene can bespecifically silenced. This could be done with skin fibroblasts orlymphocytes in vitro, or haematopoietic bone marrow stem cells. Inappropriate cases, methylated oligonucleotides could be used to silencespecific genes in vivo. These approaches resemble procedures for genetherapy, where a defective recipient gene is converted to a normal geneby uptake and integration of DNA into the chromosome. More informationcan be obtained regarding gene therapy in W. French Anderson, 1992,"Human Gene Therapy", Science 256, 808-813.

It should also be noted that the oligonucleotides used in the method ofthe present invention may hybridize to either the sense or antisensestrand of DNA.

In order to protect the single stranded oligonucleotide from degradationby nucleases it is presently preferred that methylated phosphorothioateoligonucleotides are used. Further information concerning sucholigonucleotides may be found in Eckstein & Gish, 1989, TIBS 14, 97-100.

As will be recognized by those skilled in the art the present inventionhas a wide number of applications. By silencing the histocompatibilitygenes "universal" donor cells for use in transplantation can beproduced. Indeed, it is foreseeable that a wide range of cells which donot express antigens typically involved in transplantation rejection canbe produced. This opens up the possibility of universal donor cells.

An obvious model target to prove the system is the gene coding for HPRT(hypoxanthine phosphoribosyl transferase) because it is X linked, withonly one copy in male cells and one active copy in female cells. Cellswith an active HPRT gene can grow in HAT medium (containinghypoxanthine, aminopterin and thymidine) but are killed by 6 thioguanine(6TG). Cells without HPRT are resistant to 6TG, but cannot grow in HATmedium. Thus, one can select for loss of enzyme activity followingsilencing of the gene, and also for gain of activity followingreactivation of the gene. Reactivation can be achieved by thedemethylating agent 5 azacytidine.

The human, mouse and hamster HPRT genes have been sequenced. Theycontain 9 exons and very long introns. At the 5' end of the gene is aCpG island, which is normally unmethylated, but which becomes methylatedduring X chromosome inactivation. The promoter region contains many CpGsites, but there are reasons to believe that important regulatory sitesmay be in a relatively short stretch of DNA (100-200 nucleotides).

Experiments are carried out with male human fibroblasts, strain MRC-5.Methylated oligonucleotides of 40-50 length covering different regionsof the promoter can by synthesized, and prepared for treating the cells.

These will be partially synchronized, following serum deprivation, andtreated when the majority are in S phase -20 hours after replacingserum. The cells are allowed 6-8 days expression time to allowpre-existing HPRT to disappear, and then plated on 6TG medium. Coloniesappearing will be picked, grown up, and tested for reactivation byazacytidine. Such reactivants can be selected on HAT medium. If nocolonies are obtained, oligonucleotides with other sequences in thepromoter region will be tested.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

I claim:
 1. A method of silencing a specific gene in a cell in vitrocomprising introducing into the cell a single stranded oligonucleotidecontaining 5-methyl deoxycytosine, the single stranded oligonucleotidehaving a sequence complementary to a sequence within the promoter regionof the gene to be silenced, wherein the sequence within the promoterregion contains at least one CpG doublet.
 2. A method of silencing aspecific gene in a cell in vitro comprising introducing into the cell asingle stranded oligonucleotide containing 5-methyl deoxycytosine, thesingle stranded oligonucleotide having a sequence complementary to asequence within a regulatory site of the gene to be silenced, whereinthe sequence within the regulatory site contains at least one CpGdoublet.
 3. A method of testing for silencing of a gene in a cellcomprising:(a) introducing into the cell a single strandedoligonucleotide containing 5-methyl deoxycytosine, the single strandedoligonucleotide having a sequence complementary to a sequence within thepromoter region of the gene to be silenced, wherein the sequence withinthe promoter region contains at least one CpG doublet; (b) selecting forloss of gene product activity following silencing of the gene by growthon a selective culture medium; and (c) reactivating the silenced genewith a demethylating agent.
 4. A method of testing for silencing of agene in a cell comprising:(a) introducing into the cell a singlestranded oligonucleotide containing 5-methyl deoxycytosine, the singlestranded oligonucleotide having a sequence complementary to a sequencewithin a regulatory site of the gene to be silenced, wherein thesequence within the regulatory site contains at least one CpG doublet;(b) selecting for loss of gene product activity following silencing ofthe gene by growth on a selective culture medium; and (c) reactivatingthe silenced gene with a demethylating agent.
 5. A method according toany one of claims 1, 2, 3, and 4, wherein the single strandedoligonucleotide comprises about 20-40 nucleotides.
 6. A method accordingto any one of claims 1, 2, 3, and 4, wherein the single strandedoligonucleotide is a methylated phosphorothioate oligonucleotide.
 7. Amethod according to any one of claims 1, 2, 3, and 4, wherein the cellis contained within a cell culture, which has been treated so that thecell cycles of each of the cells contained therein are synchronized. 8.A method according to claim 7, wherein the single strandedoligonucleotide is introduced to the cell when the cells containedwithin the culture are in S phase.